Despite a reduction in both incidence and mortality of colorectal cancer, partially due to rapidly increased use of endoscopy, colorectal cancer (CRC) still remains the 4th most common incident cancer and the 2nd most common cause of cancer death in the US. Thus, it is critical to develop new prevention strategies. The human colorectum is host to billions of bacteria which comprise the microbiota. The microbiota is essential in several functions of colorectal health including immunity, nutrient metabolism, growth, and energy harvesting. However, until recently, evaluation of the role of microbiota in health has been limited because the majority of bacteria are uncultivable. Cultivation-independent next-generation sequencing of 16S ribosomal RNA has permitted the evaluation of the role of microbiota in health. However, previous epidemiological studies of the colorectal microbiome are few and with small sample sizes. Furthermore, recent studies found oxygen gradients in the intestine are the steepest in the body, which reduce from mucosa to near anoxia at the middle of the lumen. Thus, microbes in luminal communities are more likely to be anaerobic compared to mucosal bacteria. Thus, before conducting large-scale epidemiological studies, it is necessary to address two key methodological or practical questions. First, what source(s) of sample(s) is/are most appropriate for analysis of the human colorectal microbiome, and, second, whether a spot sample is adequately reliable for representing the colorectal microbiome in relation to colorectal carcinogenesis. This proposed study will address these two key issues by using banked fecal samples and paired rectal swabs from 2 time points among 100 participants with a history of colorectal adenoma (4 samples from each person). In the parent study, colorectal carcinogenesis biomarkers (apoptosis, inflammation, proliferation and Wnt pathway) are measured in normal rectal tissue at the same 2 time points as the fecal and rectal swab samples. In this proposed pilot study, the samples will be analyzed using next-generation sequencing of 16S rRNA. We will compare 1) swabs to stool and 2) the combination of swab and stool to the single sample type in association with immunohistochemistry carcinogenesis biomarkers. We will also examine whether the microbial biomarkers in rectal swabs and stool samples remain stable over time; and further 2) to compare microbial biomarkers in stool and rectal swabs collected at one time point (spot collection) with microbial biomarkers from two time points (3 months apart) in their correlation with immunohistochemistry carcinogenesis biomarkers. The proposed study will not only investigate the validity of microbial biomarkers in different types or combinations of samples, but also examine the reliability of these biomarkers from one spot sample vs. collections from two time points in relation to carcinogenesis biomarkers in rectal tissue. The proposed study is unique with a large sample size, multiple sample types, and multiple collections across time points. The results from the proposed study will lay a solid foundation for future large-scale epidemiologic studies of microbiota in association with CRC.